Isopropylmalate synthase variant and a method of producing L-leucine using the same

ABSTRACT

A novel modified polypeptide having an isopropylmalate synthase activity, a polynucleotide encoding the same, a microorganism comprising the polypeptide, and a method of producing L-leucine by culturing the microorganism.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase under 35 U.S.C. § 371 ofInternational Application PCT/KR2017/011622, filed Oct. 20, 2017, whichwas published in Korean as WO 2018/124440 on Jul. 5, 2018, which claimspriority to Korean Patent Application No. 10-2016-0181343, filed Dec.28, 2016, the entire contents of which are incorporated herein byreference.

SEQUENCE LISTING STATEMENT

The present application contains a Sequence Listing, which is beingsubmitted via EFS-Web on even date herewith. The Sequence Listing issubmitted in a file entitled “Sequence Listing HAN030-009APC.txt,” whichwas created on Jun. 26, 2019, and is approximately 121 kb in size. ThisSequence Listing is hereby incorporated by reference.

TECHNICAL FIELD

The present disclosure relates to a novel modified polypeptide having anisopropylmalate synthase activity, a polynucleotide encoding the same, amicroorganism comprising the polypeptide, and a method of producingL-leucine by culturing the microorganism.

BACKGROUND ART

L-Leucine is an essential amino acid, one which is expensive and widelyused in medicaments, foods, feed additives, industrial chemicals, etc.In addition. L-leucine is mainly produced using a microorganism.Fermentation of branched-chain amino acids including L-leucine is mainlycarried out through a microorganism of the genus Escherichia or amicroorganism of the genus Corynebacterium, known to biosynthesize2-ketoisocaproate as a precursor from pyruvic acid though several steps(Korean Patent Nos. 10-0220018 and 10-0438146).

Isopropylmalate synthase (hereinafter referred to as “IPMS”), which isan enzyme involved in the biosynthesis of leucine, is an enzyme of thefirst step in the biosynthesis of leucine, which converts2-ketoisovalerate, produced during the valine biosynthetic pathway, toisopropylmalate, allowing the biosynthesis of leucine instead of valine,and thereby IPMS is an important enzyme, in the process of leucinebiosynthesis. However, the IPMS is subject to feedback inhibition byL-leucine, which is a final product, or derivatives thereof.Accordingly, although there is a variety of prior art relevant to IPMSvariants which release feedback inhibition for the purpose of producinga high concentration of leucine (U.S. Patent Publication Application No.2015-0079641 and U.S. Pat. No. 6,403,342), research is still continuingto discover better variants.

DISCLOSURE Technical Problem

The present inventors have endeavored to develop an IPMS variant thatcan be used for the production of L-leucine with a high concentration,and as a result, the present inventors developed a novel IPMS variant.It was confirmed that the variant released feedback inhibition byL-leucine, which is a final product, and enhanced an activity thereofsuch that the variant is capable of producing L-leucine at a high yieldfrom a microorganism containing the same, thereby completing the presentdisclosure.

Technical Solution

An object of the present disclosure is to provide a novel modifiedpolypeptide having an isopropylmalate synthase activity.

Another object of the present disclosure is to provide a polynucleotideencoding the modified poly peptide.

Still another object of the present disclosure is to provide amicroorganism of the genus Corynebacterium producing L-leucine,containing the polypeptide.

Still another object of the present disclosure is to provide a method ofproducing L-leucine by culturing the microorganism in a medium.

Advantageous Effects

The novel modified polypeptide having an activity of isopropylmalatesynthase is a polypeptide in which the activity is increased compared tothat of the wild-type and feedback inhibition by L-leucine is released,and thereby L-leucine can be produced in a high yield using suchmodified polypeptide.

BEST MODE FOR CARRYING OUT THE INVENTION

To achieve the above objects, an aspect of the present disclosureprovides a novel modified polypeptide having an isopropylmalate synthaseactivity. The novel modified polypeptide may be a modified polypeptidehaving an isopropylmalate synthase activity, wherein arginine atposition 558 from a N-terminus of a polypeptide consisting of the aminoacid sequence of SEQ ID NO: 1 is substituted with an amino acid residueother than arginine, or glycine at position 561 from a N-terminus of apolypeptide consisting of the amino acid sequence of SEQ ID NO: 1 issubstituted with an amino acid residue other than glycine. The modifiedpolypeptide of the present disclosure not only has an activity higherthan that of a polypeptide of SEQ ID NO: 1 having an isopropylmalatesynthase activity, but also has a feature that feedback inhibition byL-leucine is released.

As used herein, the term “isopropylmalate synthase” refers to an enzymeconverting 2-ketoisovalerate to isopropyhnalate, which is a precursor ofL-leucine, by reacting with acetyl-CoA. The isopropylmalate synthase ofthe present disclosure may be included as long as the enzyme has theconversion activity, regardless of an origin of a microorganism.Specifically, the isopropylmalate synthase may be an enzyme derived froma microorganism of the genus Corynebacterium. More specifically, theisopropylmalate synthase may be an isopropylmalate synthase derived fromCorynebacterium glutamicum, and specifically, it may include the aminoacid sequence of SEQ ID NO: 1, but is not limited thereto. Additionally,the isopropyhnalate synthase may include a polypeptide having homologyof at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% with the amino acidsequence of SEQ ID NO: 1. For example, it is obvious that an amino acidsequence having such homology and exhibiting an effect corresponding tothat of the isopropylmalate synthase can be included within the scope ofthe present disclosure even if it has an amino acid sequence in whichsome of the sequences are deleted, modified, substituted, or added.

As used herein, the term “increase in activity of isopropylmalatesynthase” refers to an increase in the conversion activity toisopropylmalate. Therefore, the modified polypeptide of the presentdisclosure has a higher level of the isopropylmalate conversion activitycompared to a polypeptide of SEQ NO: 1 having the activity ofisopropylmalate synthase. The isopropylmalate conversion activity can bedirectly confirmed by measuring the level of isopropylmalate produced,or can be indirectly confirmed by measuring the level of CoA produced.As used herein, the term “increase in activity” may be used incombination with “enhanced activity”, Further, isopropyimalate is aprecursor of L-leucine, and thus the use of the modified polypeptide ofthe present disclosure results in producing a higher level of L-leucinecompared to a polypeptide of SEQ ID NO: 1 having the activity ofisopropylmalate synthase.

Additionally, unlike a polypeptide of SEQ ID NO: 1 having the activityof isopropylmalate synthase, the modified polypeptide of the presentdisclosure may be characterized in that feedback inhibition byL-leucine, which is a final product, or a derivative thereof isreleased. As used herein, the term “feedback inhibition” refers to theinhibition of a reaction at the early state of an enzyme system by afinal product in the enzyme system. For the objects of the presentdisclosure, the feedback inhibition may be feedback inhibition in whichL-leucine or a derivative thereof inhibits the activity ofisopropylmalate synthase, which mediates the first step of thebiosynthetic pathway, but is not limited thereto. Therefore, when thefeedback inhibition of isopropylmalate synthase is released, theproductivity of L-leucine can be increased compared with the case of notreleasing the same.

As used herein, the term “modification”, “modified”, or “variant” refersto a culture or an individual that shows an inheritable or non-heritablealternation in one stabilized phenotype. Specifically, the term“variant” may be intended to mean a variant in which its activity isefficiently increased because the amino acid sequence corresponding toCorynebacterium glutamicum-derived isopropylmalate synthase is modifiedcompared to the wild-type, a variant in which feedback inhibition byL-leucine or a derivative thereof is released, or a variant in which theincrease in activity and feedback inhibition are both released.

Specifically, the modified polypeptide of the present disclosure, whichhas the activity of isopropylmalate synthase, may be a modifiedpolypeptide having an activity of isopropylmalate synthase, whereinarginine, an amino acid at position 558 from a N-terminus of apolypeptide consisting of the amino acid sequence of SEQ ID NO: 1, issubstituted with an amino acid residue other than arginine, or glycine,an amino acid residue at position 561 from a N-terminus of a polypeptideconsisting of the amino acid sequence of SEQ ID NO: 1, is substitutedwith an amino acid residue other than glycine. The amino acid other thanarginine may include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, methionine, glycine, serine, threonine,cysteine, tyrosine, asparagine, glutamine, lysine, histidine, asparticacid, and glutamic acid; and the amino acid other than glycine mayinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, methionine, arginine, serine, threonine, cysteine, tyrosine,asparagine, glutamine, lysine, histidine, aspartic acid, and glutamicacid; but the amino acids are not limited thereto. More specifically,the modified polypeptide may be a modified polypeptide, whereinarginine, an amino acid residue at position 558 from a N-terminus of apolypeptide consisting of the amino acid sequence of SEQ ID NO: 1, issubstituted with histidine, alanine, or glutamine; or glycine, an aminoacid residue at position 561 from a N-terminus of a polypeptideconsisting of the amino acid sequence of SEQ ID NO: 1, is substitutedwith aspartic acid, arginine, or tyrosine, but is not limited thereto.Additionally, the modified polypeptide may be one in which the arginineat position 558 is substituted with histidine, alanine, or glutamine;and the glycine at position 561 is substituted with aspartic acid,arginine, or tyrosine, but is not limited thereto. Most specifically,the modified polypeptide may include an amino acid sequence of any oneof SEQ ID NO: 21 to SEQ ID NO: 35.

Additionally, the modified polypeptide may include a polypeptide havinghomology of at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% with the aminoacid sequence of any one of SEQ ID NO: 21 to SEQ ID NO: 35. For example,it is obvious that an enzyme variant having an amino acid sequence, inwhich some of the sequences are deleted, modified, substituted, or addedwhile the modified amino acid sequence corresponding to the amino acidsequence at positions 558 and/or 561 is fixed, should also belong to thescope of the present disclosure as long as the amino acid sequence hasthe homology above and exhibits an effect corresponding to that ofisopropylmalate synthase. On the other hand, positions 558 and 561,which are specific modification positions, refer to positions that aredetermined based on the N-terminus in the amino acid sequence of SEQ IDNO: 1, and therefore, the fact that such positions are determined byconsidering the number of the amino acids which are added to or deletedfrom the N-terminus of SEQ ID NO: 1 is obvious to one of ordinary skillin the art, and thereby also belongs to the scope of the presentdisclosure. For example, leuA, which is the gene encodingisopropylmalate synthase, was represented by SEQ ID NO: 1 consisting of616 amino acids. However, in some references, the translation initiationcodon is indicated 35 amino acids downstream of the sequence of the leuAgene, i.e., a gene consisting of 581 amino acids. In such a case, the558t^(h) amino acid is interpreted as the 523rd amino acid and the 561stamino acid as the 526t^(h) amino acid, and is thereby included in thescope of the present disclosure.

As used herein, the term “homology” refers to a percentage of identitybetween two polynucleotides or polypeptide moieties. The homologybetween sequences from a moiety to another moiety may be determined bythe technology known in the art. For example, the homology may bedetermined by directly arranging the sequence information, i.e.,parameters such as score, identity, similarity, etc., of twopolynucleotide molecules or two polypeptide molecules using an easilyaccessible computer program (Example: BLAST 2.0). Additionally, thehomology between polynucleotides may be determined by hybridizingpolynucleotides under the condition of forming a stable double-strandbetween the homologous regions, disassembling with a singlestrand-specific nuclease, followed by size determination of thedisassembled fragments.

Another aspect of the present disclosure provides a polynucleotideencoding the modified polypeptide.

The polynucleotide may be a polynucleotide encoding a modifiedpolypeptide having the activity of isopropylmalate synthase, whereinarginine, an amino acid at position 558 from a N-terminus of apolypeptide consisting of the amino acid sequence of SEQ ID NO: 1, issubstituted with another amino acid residue other than arginine, orglycine, an amino acid residue at position 561 from a N-terminus of apolypeptide consisting of the amino acid sequence of SEQ ID NO: 1, issubstituted with another amino acid residue other than glycine.Specifically, a polynucleotide encoding a polypeptide including theamino acid sequence of SEQ ID NOs: 21 to 35 and having an activity ofisopropylmalate synthase; a modified polypeptide having homology of atleast 80%, 90%, 95%, 96%, 97%, 98%, or 99% with the polypeptide above;or encoding a modified polypeptide having an activity of isopropylmalatesynthase, in which some of the sequences are deleted, modified,substituted, or added while the modified amino acid sequence atpositions 558 and/or 561, which are specific modification positions inthe polypeptide above, is fixed may be included without limitation.Alternately, the probe that can be prepared from a known gene sequence,for example, a sequence encoding a protein having activity ofIsopropylmalate synthase by hybridization of a complementary sequencefor all or part of the nucleotide sequence above under stringentconditions, can be included without limitation.

As used herein, the term “stringent conditions” refers to conditionsunder which a so-called hybrid is formed while non-specific hybrids arenot formed. Examples of such conditions include conditions under whichgenes having high degrees of homology, such as genes having a homologyof 80% or more, specifically 90% or more, more specifically 95% or more,furthermore specifically 97% or more, and most specifically 99% or more,hybridize with each other while genes having low degrees of a homologydo not hybridize with each other, or conditions under which genes arewashed 1 time, and specifically 2 and 3 times, at a temperature and asalt concentration equivalent to 60° C., 1× SSC, and 0.1% SDS,specifically 60° C., 0.1× SSC, and 0.1% SDS, and more specifically 68°C., 0.1× SSC, and 0.1% SDS, which are the conditions for washing ofordinary Southern hybridization (Sambrook et al., Molecular Cloning: ALaboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2001)).

The probe used in the hybridization may be a part of the complementarysequence of the nucleotide sequence. Such probe can be constructed byPCR using an oligonucleotide prepared based on a known sequence as aprimer and a gene fragment containing such nucleotide sequence as atemplate. For example, a gene fragment having a length of about 300 bpcan be used as a probe. More specifically, in the case of using a probehaving a length (about 300 bp), 50° C., 2× SSC, and 0.1% SDS may besuggested for the washing conditions of hybridization.

On the other hand, the polynucleotide may be a polynucleotide having anucleotide sequence of any one of SEQ ID NO: 36 to SEQ ID NO: 50, and itis obvious that the polynucleotide also includes a polynucleotide thatcan be translated into the modified polypeptide by codon degeneracy.

Still another aspect of the present disclosure is to provide amicroorganism of the genus Corynebacterium producing L-leucine,containing the modified polypeptide.

In the present disclosure, the microorganism may include all of amicroorganism artificially produced through transformation or anaturally-occurring microorganism.

As used herein, the term “transformation” refers to the introduction ofa gene into a host cell for expression. In the present disclosure, thetransformation method includes any method that introduces a gene into acell and can be carried out by selecting a suitable standard techniqueknown in the art. Examples of the transformation method areelectroporation, calcium phosphate co-precipitation, retroviralinfection, microinjection, DEAE-dextran, cationic liposome, heat shockmethod, etc., but are not limited thereto.

The gene to be transformed may include both a form inserted into thechromosome of a host cell and a form located outside the chromosome, aslong as it can be expressed in the host cell. In addition, the geneincludes DNA and RNA as a polynucleotide capable of encoding apolypeptide, and any gene that can be introduced and expressed in thehost cell can be used without limitation. For example, the gene can beintroduced into a host cell in a form of an expression cassette, whichis a polynucleotide construct containing all elements required forself-expression. The expression cassette usually includes a promoteroperably linked to the gene, a transcription termination signal,ribosome binding sites, and a translation termination signal. Theexpression cassette may be in a form of a self-replicable expressionvector. In addition, the gene may be one introduced into a host cellitself or in a form of a polynucleotide construct, i.e., a form of avector, and operably linked to the sequences required for expression inthe host cell.

As used herein, the term “vector” refers to any carrier for cloningand/or transferring nucleotides to a host cell. A vector may be areplicon to allow for the replication of the fragments combined withother DNA fragments. “Replicon” refers to any genetic unit acting as aself-replicating until for DNA replication in vivo, that is, replicableby self-regulation (e.g., plasmid, phage, cosmid, chromosome, andvirus). The term “vector” may include viral and non-viral carriers forintroducing nucleotides into a host cell in vitro, ex vivo, or in vivo,and may also include a mini-spherical DNA. For example, the vector maybe a plasmid without a bacterial DNA sequence. Removal of bacterial DNAsequences which are rich in CpG area has been conducted to reducesilencing of the transgene expression and to promote more continuousexpression from a plasmid DNA vector (for example, Ehrhardt, A. et al.(2003) Hum Gene Ther 10: 215-25; Yet, N. S. (2002) MoI Ther 5: 731-38;Chen, Z. Y. et al. (2004) Gene Ther 11: 856-64). The term “vector” alsomay include a transposon such as Sleeping Beauty (Izsvak et al. J. Mol.Biol. 302:93-102 (2000)), or an artificial chromosome. Examples of thevector typically used may be natural or recombinant plasmid, cosmid,virus, and bacteriophage. For example, as the phage vector or the cosmidvector, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λ11,Charon4A, Charon21A, etc. may be used. In addition, as the plasmidvector, pDZ type, pBR type, pUC type, pBluescriptII type, pGEM type, pTZtype, pCL type, pET type, etc. may be used. Specifically, pECCG117vector may be used. The vector that can be used in the presentdisclosure is not particularly limited, and the knownexpression/substitution vector may be used.

In addition, the vector may be a recombinant vector which may furtherinclude various antibiotic resistance genes.

As used herein, the term “antibiotic resistance gene” refers to a genehaving resistance to antibiotics, and the cells comprising this genesurvive even in the environment treated with the correspondingantibiotic. Therefore, the antibiotic resistance gene can be effectivelyused as a selection marker for a large-scale production of plasmids inmicroorganisms, such as E. coli, etc. In the present invention, as theantibiotic resistance gene is not a factor that significantly affectsthe expression efficiency which is obtained by an optimal combination ofcomponents of the vector which is the key feature of the presentinvention, any common antibiotic resistance gene can be used as aselection marker without limitation. Specifically, the resistance genesagainst ampicilin, tetracyclin, kanamycin, chloramphenicol,streptomycin, or neomycin can be used.

As used herein, the term “operably linked” refers to the operablelinking of a regulatory sequence for nucleotide expression with anucleotide sequence encoding a target protein for performing its generalfunction, thereby affecting the expression of a coding nucleotidesequence. Operable linking with a vector can be made using a generecombination technique known in the art, and site-specific DNA cleavageand ligation can be performed using a restriction enzyme and ligaseknown in the art.

As used herein, the term “host cell in which a vector is introduced(transformed)” refers to a cell transformed with a vector having a geneencoding one or more target proteins. The host cell may include any of aprokaryotic microorganism and a eukaryotic microorganism as long as themicroorganism includes a modified polypeptide capable of producingisopropylmalate synthase by introducing the vector above. For example,the microorganism strain belonging to the genera of Escherichia,Erwinia, Serratia, Providencia, Corynebacterium, and Brevibacterium maybe included. An example of the microorganism of the genusCorynebacterium may be Corynebacterium glutamicum, but is not limitedthereto.

The microorganism of the genus Corynebacterium producing L-leucine,which is capable of expressing the modified polypeptide having theactivity of isopropylmalate synthase, includes all microorganismscapable of expressing the modified polypeptide by various known methodsin addition to the introduction of a vector.

Still another aspect of the present disclosure provides a method ofproducing L-leucine, comprising: (a) culturing the microorganism of thegenus Corynebacterium producing L-leucine; and (b) recovering L-leucinefrom the cultured microorganism or the cultured medium.

As used herein, the term “culture” refers to culturing of themicroorganism under appropriately controlled environmental conditions.The culturing process of the present disclosure may be carried outdepending on a suitable medium and culture condition known in the art.Such culturing process can be easily adjusted and used by one ofordinary skill in the art depending on the strain to be selected.Specifically, the culture may be a batch type, a continuous type, and afed-batch type, but is not limited thereto.

The carbon sources contained in the medium may include sugars andcarbohydrates, such as glucose, sucrose, lactose, fructose, maltose,starch, and cellulose; oils and fats, such as soybean oil, sunfloweroil, castor oil, coconut oil, etc.; fatty acids, such as palmitic acid,stearic acid, and linoleic acid; alcohols, such as glycerol and ethanol;and organic acids such as acetic acid. These materials may be used aloneor in combinations thereof, but are not limited thereto. The nitrogensources contained in the medium may include organic nitrogen sources,such as peptone, yeast extract, gravy, malt extract, corn steep liquor,and soybean; and inorganic nitrogen sources, such as urea, ammoniumsulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, andammonium nitrate. These nitrogen sources may be used alone or incombinations thereof, but are not limited thereto. The phosphoroussources contained in the medium may include potassium dihydrogenphosphate, dipotassium hydrogen phosphate, and correspondingsodium-containing salts, but are not limited thereto. Additionally,metal salts such as magnesium sulfate or iron sulfate may be contained.In addition, amino acids, vitamins, suitable precursors, etc. may becontained. These media or precursors may be added in a batch cultureprocess or a continuous culture process to a culture, but are notlimited thereto.

pH of the culture may be adjusted during the cultivation by adding anappropriate compound such as ammonium hydroxide, potassium hydroxide,ammonia, phosphoric acid, and sulfuric acid, and the generation of foamsmay be inhibited during the cultivation by using an antifoaming agentsuch as fatty acid polyglycol ester. In order to maintain aerobicconditions of the culture, oxygen or oxygen-containing gas may beinjected into the culture. In order to maintain anaerobic andmicroaerobic conditions, no gas may be injected or nitrogen, hydrogen,or carbon dioxide may be injected. The temperature of the culture may be27° C. to 37° C., and specifically 30° C. to 35° C., but is not limitedthereto. The period of cultivation may be continued as long as thedesired amount of useful material is recovered, and preferably for 10 to100 hours, but the period of cultivation is not limited thereto.

The step of recovering L-leucine produced in the culture step of thepresent disclosure can collect the desired L-leucine from themicroorganism or the medium using a suitable method known in the artdepending on culture methods. For example, centrifugation, filtration,anion exchange chromatography, crystallization, HPLC, etc. may be used,and a suitable method known in the art may be used to recover thedesired L-leucine from the medium or the microorganism. Additionally,the recovery step above may include a purification process.

Mode for Carrying Out the Invention

Hereinbelow, the present disclosure will be described in detail withaccompanying exemplary embodiments. However, the exemplary embodimentsdisclosed herein are only for illustrative purposes and should not beconstrued as limiting the scope of the present disclosure.

EXAMPLE 1 Confirmation of leuA Nucleotide Sequence of KCCM11661P,Microorganism Producing Leucine

Corynebacterium glutamicum ATCC14067 was inoculated into a seed mediumhaving the ingredients described below at 121° C. for 15 minutes,cultured for 13 hours, and then 25 mL of the culture medium wasrecovered. The recovered culture medium was washed with a 100 mM citratebuffer and treated with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) for30 minutes to a final concentration of 400 μg/mL. Thereafter, theresultant was washed with a 100 mM phosphate buffer. The mortality rateof the strains treated with NTG was determined to be 99.6% as a resultof smearing the strains on a minimal medium having the ingredientsdescribed below. In order to achieve variants resistant to norleucine(NL), the NTG-treated strains were smeared on the minimal media withfinal concentrations of 20 mM, 40 mM, and 50 mM, cultured at 30° C. for5 days, and then variants resistant to NL were obtained.

Seed Medium

Glucose (20 g), peptone (10 g), yeast extract (5 g), carbamide (1.5 g),KH2PO4 (4 g), K2HPO4 (8 g), MgSO₄.7H₂O (0.5 g), biotin (100 μg),thiamine hydrochloride (1,000 μg), calcium-pantothenic acid (2,000 μg),nicotinamide (2,000 μg; based on 1 liter of distilled water), pH 7.0<Production medium >

Glucose (100 g), (NH₄)₂SO₄ (40 g), soy protein (2.5 g), corn steep solid(5 g), urea (3 g), KH₂PO₄ (1 g), MgSO₄.7H₂O (0.5 g), biotin (100 μg),thiamine hydrochloride (1,000 μg), calcium-pantothenic acid (2000 μg),nicotinamide (3,000 μg), CaCO₃ (30 g; based on 1 liter of distilledwater), pH 7.0

The variants obtained by the method above were designated asCorynebacterium glutamicum KCJ-24 and Corynebacterium glutamicum KCJ-28and deposited to the Korean Culture Center of Microorganisms, aninternational depositary authority, on Jan. 22, 2015, under the BudapestTreaty, and as a result, Corynebacterium glutamicum KCJ-24 andCorynebacterium glutamicum KCJ-28 were deposited under Accession Nos.KCCM11661P and KCCM11662P, respectively. Corynebacterium glutamicumKCJ-24 and Corynebacterium glutamicum KCJ-28 produced L-leucine at aconcentration of 2.7 g/L and 3.1 g/L, respectively. Therefore, it wasconfirmed that the productivity of L-leucine produced from the variantswas 10-fold higher than that of the wild-type.

Additionally, an attempt was made to confirm whether the variation ofleuA encoding isopropylmalate synthase (IPMS) occurred in the variantKCCM11661P. The amino acid sequence (SEQ ID NO: 1) of wild-type leuA wasconfirmed by referring to WP_003863358.1 of Genebank. The chromosomalDNA of the variant was amplified using a polymerase chain reaction(hereinafter referred to as ‘PCR’) method. Although it is known that theleuA gene consists of 616 amino acids, in some references, it ispublished that the translation initiation codon is indicated 35 aminoacids downstream of the sequence of the leuA gene, and thereby the leuAgene consists of 581 amino acids. In such a case, the position numberindicating the variation of the corresponding amino acid can vary.Therefore, in cases where the leuA gene is considered to consist of 581amino acids, the variation position is additionally indicated inparenthesis.

Specifically, PCR was performed using the chromosomal DNA of the variantas a template and using primers of SEQ ID NOs: 3 and 4 under thefollowing conditions: denaturation at 94° C. for 1 minute; annealing at58° C. for 30 seconds; and polymerization at 72° C. for 2 minutes usingTaq DNA polymerase. Such PCR was repeated a total of 28 times to amplifya fragment of about 2,700 base pairs. The nucleotide sequence of thefragment was analyzed using the same primer, and as a result, it wasconfirmed that G, which is the 1673nd nucleotide of leuA in KCCM11661P,was substituted with A. This result implies that arginine, which is the558^(th) (or 523^(rd); hereinafter only indicated as 558^(th)) aminoacid, is substituted with histidine. In addition, it was also confirmedthat GC, which are the 1682^(nd) and 1683^(rd) nucleotides, weresubstituted with AT. This result also implies that glycine, which is the561^(st) (or 526^(th), hereinafter only indicated as 561^(st)) aminoacid, is substituted with aspartic acid.

EXAMPLE 2 Production of Substitution Vector of IPMS Variant

In order to produce a vector containing the modified nucleotide sequenceconfirmed in Example 1, PCR was performed using the chromosomal DNA ofthe variant above as a template and using primers of SEQ ID NOs: 5 and 6under the following conditions: denaturation at 94° C. for 1 minute;annealing at 58° C. for 30 seconds; and polymerization at 72° C. for 1minute using Pfu DNA polymerase. Such PCR was repeated a total of 25times to amplify a fragment of about 1,460 base pairs with SalI and XbaIrestriction enzyme sites. The amplified fragment was treated withrestriction enzymes, SalI and XbaI, and then pDZ-leuA (R558H, G561D) wasprepared by ligation with the vector pDZ (Korean Patent No: 10-0924065and International Patent Publication No. 2008-033001) treated with thesame enzymes. Additionally, in order to prepare a vector with eachvariation, ATCC14067 was used as a template, and then 2 fragments wereamplified using primers 5 and 7, and primers 8 and 6, respectively. PCRwas performed using the two prepared fragments as templates under thefollowing conditions: denaturation at 94° C. for 1 minute; annealing at58° C. for 30 seconds; and polymerization at 72° C. for 1 minute usingPfu DNA polymerase. Such PCR was repeated a total of 25 times to amplifya fragment of about 1,460 base pairs with SalI and XbaI restrictionenzyme sites. The amplified fragment was treated with restrictionenzymes SalI and XbaI, and then pDZ-leuA (R558H) was prepared byligation with pDZ treated with the same enzymes. pDZ-leuA (G561D) wasprepared using primers 5 and 9, and primers 10 and 6 by the same methodabove.

EXAMPLE 3 Production of Substitution Strain of IPMS Variant

Corynebacterium glutamicum ATCC14067 was used as a parent strain inorder to prepare a strain containing the leuA-modified nucleotidesequence which was found in the modified strain above.

Corynebacterium glutamicum ATCC14067 was transformed with the vectorspDZ-leuA (R558H), pDZ-leuA (G561D), and pDZ-leuA (R558H, G561D), whichwere prepared in Example 2 by electroporation. Each of the strainsprepared through the secondary crossover was designated as 14067::leuA(R558H), 14067::leuA (G561D), and 14067::leuA (R558H, G561D). In orderto confirm whether the nucleotide of leuA was substituted, PCR wasperformed using primers of SEQ ID NOs: 3 and 4 under the followingconditions: denaturation at 94° C. for 1 minute; annealing at 58° C. for30 seconds; and polymerization at 72° C. for 2 minutes using Taq DNApolymerase. Such PCR was repeated a total of 28 times to amplify afragment of about 2,700 base pairs. Thereafter, the substitution of thenucleotide of leuA was confirmed by analyzing the nucleotide sequencewith the same primer.

The strain, 14067::leuA (R558H, G561D) which was transformed with thevector pDZ-leuA (R558H, G561D), was designated as KCJ-0148, anddeposited to the Korean Culture Center of Microorganisms on Jan. 25,2016, and as a result, the strain was deposited under Accession No.KCCM11811P.

EXAMPLE 4 Production of L-Leucine in Substitution Strain of IPMS Variant

In order to produce L-leucine from Corynebacterium glutamicum14067::leuA (R558H), 14067::leuA (G561D), and 14067::leuA (R558H,G561D), which were prepared in Example 3, cultivation was carried out inthe following manner.

A platinum loop of each of the parent strain, Corynebacterium glutamicumATCC14067, and the prepared Corynebacterium glutamicum 14067::leuA(R558H), 14067::leuA (G561D), and 14067::leuA (R558H, G561D) strains wasinoculated into a corner-baffled flask (250 mL) containing a productionmedium (25 mL). Thereafter, L-leucine was produced by incubating in ashaking water bath at 30° C. at a rate of 200 rpm for 60 hours.

After completion of the incubation, the amount of L-leucine produced wasmeasured by high performance liquid chromatography. The concentration ofL-leucine in the culture medium for each experimental strain is shown inTable 1 below.

TABLE 1 Production of L-leucine in substitution strain of IPMS variantL-Leucine Strain concentration (g/L) ATCC14067 0.1 14067::leuA (R558H)1.2 14067::leuA (G561D) 1.6 14067::leuA (R558H, G561D) 2.5

As shown in Table 1 above, it was confirmed that the L-leucineproductivity of the L-leucine-producing strains, Corynebacteriumglutamicum 14067::leuA (R558H), 14067::leuA (G561D), and 14067::leuA(R558H, G561D), which have the R558H, G561D, or R558H/G561D variation inthe leuA gene, was enhanced about 12- to 25-fold compared to that of theparent strain, Corynebacterium glutamicum ATCC14067.

EXAMPLE 5 Production of IPMS Variant-Overexpressing Vector

In order to produce an expression vector containing the modifiednucleotide sequence confirmed in Example 1, PCR was carried out usingATCC14067 and the chromosomal DNA of the 3 variants prepared in Example3 as templates and using primers of SEQ ID NOs: 11 and 12 under thefollowing conditions: denaturation at 94° C. for 1 minute; annealing at58° C. for 30 seconds; and polymerization at 72° C. for 1 minute usingPfu DNA polymerase. Such PCR was repeated a total of 25 times to amplifya fragment of about 2,050 base pairs with NdeI and XbaI restrictionenzyme sites. The amplified fragment was treated with restrictionenzymes, NdeI and XbaI, and then expression vectors p117_PCJ7-leuA (WT),p117_PCJ7-leuA (R558H), p117_PCJ7-leuA (G561D), and p117_PCJ7-leuA(R558H, G561D) were prepared by ligation using p117_PCJ7 in which a PCJ7promoter was inserted in the vector pECCG117 (Biotechnology letters Vol.13, No. 10, p. 721-726 (1991)) treated with the same enzymes. The PCJ7promoter is a promoter that enhances gene expression, and is publiclyknown in Korean Patent No. 10-0620092 and International PatentPublication No. 2006-065095.

EXAMPLE 6 Production of Strain Transformed With IPMSVariant-Overexpressing Vector

In order to produce a strain transformed with an overexpression vectorcontaining the leuA modified nucleotide sequence prepared in Example 5,the parent strain, which is wild-type Corynebacterium glutamicumATCC14067, and the leucine-producing strains KCCM11661P and KCCM11662Pwere used.

Each of the vectors p117_PCJ7-leuA (WT), p117_PCJ7-leuA (R558H),p117_PCJ7-leuA (G561D), and p117_PCJ7-leuA (R558H, G561D), prepared inExample 5, was transformed with Corynebacterium glutamicum ATCC14067,KCCM11661P, and KCCM11662P by electroporation. As a result,14067::p117_PCJ7-leuA (WT), 14067::p117_PCJ7-leuA (R558H),14067::p117_PCJ7-leuA (G561D), 14067::p117_PCJ7-leuA (R558H,G561D);KCCM11661P::p117_PCJ7-leuA (WT), KCCM11661P::p117_PCJ7-leuA (R558H),KCCM11661P::p117_PCJ7-leuA (G561D), KCCM11661P::p117_PCJ7-leuA (R558H,G561D); and KCCM11662P::p117_PCJ7-leuA (WT), KCCM11662P::p117_PCJ7-leuA(R558H), KCCM11662P::p117_PCJ7-leuA (G561D), KCCM11662P::p117_PCJ7-leuA(R558H, G561D) were produced.

EXAMPLE 7 Production of L-Leucine in Strain Transformed With IPMSVariant-Overexpressing Vector

In order to produce L-leucine from the L-leucine-producing strains,Corynebacterium glutamicum 14067::p117_PCJ7-leuA (WT),14067::p117_PCJ7-leuA (R558H), 14067::p117_PCJ7-leuA (G561D),14067::p117_PCJ7-leuA (R558H, G561D); KCCM11661P:: p117_PCJ7-leuA (WT),KCCM11661P::117PCJ7-leuA (R558H), KCCM11661P::p117_PCJ7-leuA (G561D),KCCM11661P::p117_PCJ7-leuA (R558H, G561D); andKCCM11662P::p117_PCJ7-leuA (WT), KCCM11662P::p117_PCJ7-leuA (R558H),KCCM11662P::p117_PCJ7-leuA (G561D), KCCM11662P::p117_PCJ7-leuA (R558H,G561D), which were produced in Example 6, cultivation was carried out inthe following manner.

A platinum loop of each of the parent strains, Corynebacteriumglutamicum ATCC14067, KCCM11661P, and KCCM11662P, and the strainsproduced in Example 6 was inoculated into a corner-baffled flask (250mL) containing a production medium (25 mL). Thereafter, L-leucine wasproduced by incubating in a shaking water bath at 30° C. at a rate of200 rpm for 60 hours.

After completion of the incubation, the amount of L-leucine produced wasmeasured by high performance liquid chromatography. The concentration ofL-leucine in the culture medium for each experimental strain is shown inTable 2 below.

TABLE 2 Production of L-leucine in strain overexpressing IPMS variantL-Leucine Strain concentration (g/L) ATCC14067 0.1 14067::p117_PCJ7-leuA (WT) 0.3 14067:: p117_PCJ7-leuA (R558H) 4.514067::p117_PCJ7-leuA (G561D) 5.1 14067::p117_PCJ7-leuA (R558H, G561D)9.8 KCCM11661P 2.7 KCCM11661P:: p117_PCJ7-leuA (WT) 3.0 KCCM11661P::p117_PCJ7-leuA (R558H) 6.1 KCCM11661P::p117_PCJ7-leuA (G561D) 6.8KCCM11661P::p117_PCJ7-leuA 12.3 (R558H,G561D) KCCM11662P 3.1KCCM11662P:: p117_PCJ7-leuA (WT) 3.3 KCCM11662P:: p117_PJ7-leuA (R558H)6.3 , KCCM11662P::p117_PCJ7-leuA (G561D) 6.9 KCCM11662P::p117_PCJ7-leuA13.1 (R558H, G561D)

As shown in Table 2 above, it was confirmed that the L-leucineproduction of the L-leucine-producing strains, 14067::p117_PCJ7-leuA(R558H), 14067::p117_PCJ7-leuA (G561D), and 14067::p117_PCJ7-leuA(R558H, G561D), which were transformed with the overexpression vectorcontaining variation of the leuA gene in the strain ATCC14067, wasenhanced 45- to 98-fold compared to that of the parent strain ATCC14067;the L-leucine production of the L-leucine-producing strains,KCCM11661P::p117_PCJ7-leuA (R558H), KCCM11661P::p117_PCJ7-leuA (G561D),and KCCM11661P::p117_PCJ7-leuA (R558H, G561D), which were transformedwith the overexpression vector containing variation of the leuA gene inthe strain KCCM11661P, was enhanced 2.3- to 4.5-fold compared to that ofthe parent strain KCCM11661P; and that the L-leucine production of theL-leucine-producing strains, KCCM11662P::p117_PCJ7-leuA (R558H),KCCM11662P::p117_PCJ7-leuA (G561D), and KCCM11662P::p117_PCJ7-leuA(R558H,G561D), which were transformed with the overexpression vectorcontaining variation of the leuA gene in the strain KCKCM11662P, wasenhanced 2- to 4.2-fold compared to that of the parent strainKCCM11662P.

EXAMPLE 8 Measurement of Isopropylmalate Synthase Activity in StrainTransformed With leua-Overexpressing Vector

In order to measure an isopropylmalate synthase activity in theL-leucine-producing strains, Corynebacterium glutamicum14067::p117_PCJ7-leuA (WT), 14067::p117_PCJ7-leuA (R558H),14067::p117_PCJ7-leuA (G561D), and 14067::p117_PCJ7-leuA (R558H, G561D),produced in Example 6, experiments were carried out in the followingmanner.

A platinum loop of each of the 4 strains above was inoculated into acorner-baffled flask (250 mL) containing the seed medium (25 mL).Thereafter, the resultants were incubated in a shaking water bath at 30°C. at a rate of 200 rpm for 16 hours. After completion of theincubation, the culture medium was centrifuged to discard thesupernatant, the pellet was washed and mixed with a lysis buffer, andthe cells were pulverized with a bead homogenizer. The proteins presentin the lysate were quantitated according to the Bradford assay, and theactivity of isopropylmalate synthase was measured by measuring the CoAproduced when the lysate containing proteins (100 μg/mL) was used. Themeasurement results of the isopropylmalate synthase activity in eachstrain are shown in Table 3 below.

TABLE 3 Strain Relative IPMS activity (%) 14067::p117_PCJ7-leuA (WT) 10014067::p117_PCJ7-leuA (R558H) 105 14067::p117_PCJ7-leuA (G561D) 13014067::p117_PCJ7-leuA (R558H, G561D) 328

In order to confirm the degree of release of feedback inhibition byleucine in the enzyme, the isopropylmalate synthase activity wasmeasured by measuring the CoA produced when the lysate containingproteins (100 μg/mL) was used under the condition where leucine (3 g/L)was added. The measurement results of the isopropylmalate synthaseactivity in each strain are shown in Table 4 below.

TABLE 4 Leucine Leucine 0 g/L 2 g/L Strain Relative IPMS activity (%)14067::p117_PCJ7-leuA (WT) 100 24 14067::p117_PCJ7-leuA (R558H) 100 6114067::p117_PCJ7-leuA (G561D) 100 70 14067::p117_PCJ7-leuA (R558H,G561D) 100 89

As shown in Tables 3 and 4 above, it was confirmed that theisopropylmalate synthase activity of the L-leucine-producing strains,Corynebacterium glutamicum 14067::p117_PCJ7-leuA (R558H),14067::p117_PCJ7-leuA (G561D), and 14067::p117_PCJ7-leuA (R558H, G561D),which were transformed with the vector expressing the IPMS variant, wereenhanced 1.05-fold, 1.3-fold, and 3.2-fold, respectively, compared tothat of the control, Corynebacterium glutamicum 14067::p117_PCJ7-leuA(WT). In addition, the L-leucine-producing strains maintained their IPMSactivity at 61%, 70%, and 89%, respectively, even when leucine (2 g/L)was added, confirming that feedback inhibition by leucine was released.

EXAMPLE 9 Production of Vector for Improving Isopropylmalate Synthase(IPMS) Variant

In Examples 4, 7, and 8, since it was confirmed that the 558t^(h) and561st amino acids in the amino acid sequence (SEQ ID NO: 1) ofisopropylmalate synthase were important sites for the activity of theIPMS variant enzyme, the attempt was made to confirm whether the enzymeactivity was enhanced or whether feedback inhibition was furtherreleased when substituted with an amino acid other than the amino acidsin the variant. Therefore, an attempt was made to prepare a variantsubstituted with an amino acid of other amino acid groups capable ofcausing structural variations.

A variant in which the 558^(th) amino acid, arginine, was substitutedwith alanine (Ala) or glutamine (Gln) was prepared. The vectorp117_PCJ7-leuA (R558A), in which the 558^(th) amino acid is substitutedwith alanine (Ala), and the vector p117_PCJ7-leuA (R558Q), in which the558^(th) amino acid is substituted with glutamine (Gln), were preparedusing a site-directed mutagenesis method and by using the vectorp117_PCJ7-leuA (R558H) as a template, the primer of SEQ ID NOs: 13 and14, and the primer pair of SEQ ID NOs: 15 and 16.

A variant in which the 561^(st) amino acid, glycine, was substitutedwith arginine (Arg) or tyrosine (Tyr) was prepared. The vectorp117_PCJ7-leuA (G561R), in which the 561^(st) amino acid is substitutedwith arginine (Arg), and the vector p117_PCJ7-leuA (G561Y), in which the561^(st) amino acid is substituted with tyrosine (Tyr), were obtainedusing a site-directed mutagenesis method and by using p117_PCJ7-leuA(G561D) as a template, the primer of SEQ ID NOs: 17 and 18, and theprimer pair of SEQ ID NOs: 19 and 20.

EXAMPLE 10 Production of Strain in Which Isopropylmalate-ModifiedVariant is Introduced

In order to prepare a strain transformed with an expression vectorcontaining the leuA-modified nucleotide sequence prepared in Example 9,wild-type Corynebacterium glutamicum ATCC14067 was used as a parentstrain.

Each of the vectors, p117_PCJ7-leuA (R558A), p117_PCJ7-leuA (R558Q),p117_PCJ7-leuA (G561R), and p117_PCJ7-leuA (G561Y), which were preparedin Example 9, was transformed in Corynebacterium glutamicum ATCC14067 byelectroporation to prepare 14067::p117_PCJ7-leuA (R558A),14067::p117_PCJ7-leuA (R558Q), 14067::p117_PCJ7-leuA (G561R), and14067::p117_PCJ7-leuA (G561Y).

EXAMPLE 11 Production of L-Leucine in Strain in Which IsopropylmalateSynthase-Modified Variant is Introduced

In order to produce L-leucine from the L-leucine-producing strains,Corynebacterium glutamicum 14067::p117_PCJ7-leuA (R558A),14067::p117_PCJ7-leuA (R558Q), 14067::p117_PCJ7-leuA (G561R), and14067::p117_PCJ7-leuA (G561Y), which were prepared in Example 10,cultivation was carried out in the following manner.

A platinum loop of each of the parent strain, Corynebacterium glutamicumATCC14067, and the 4 strains above was inoculated into a corner-baffledflask (250 mL) containing a production medium (25 mL). Thereafter,L-leucine was produced by incubating in a shaking water bath at 30° C.at a rate of 200 rpm for 60 hours.

After completion of the incubation, the amount of L-leucine produced wasmeasured by high performance liquid chromatography. The concentration ofL-leucine in the culture medium for each experimental strain is shown inTable 5 below.

TABLE 5 Production of L-leucine in strain overexpressing IPMS variantL-Leucine Strain concentration (g/L) ATCC14067 0.1 14067::p117_PCJ7-leuA(WT) 0.3 14067::p117_PCJ7-leuA (R558H) 4.5 (Example 7)14067::p117_PCJ7-leuA (R558A) 3.8 14067::p117_PCJ7-leuA (R558Q) 3.214067::p117_PCJ7-leuA (G561D) 5.1 (Example 7) 14067::p117_PCJ7-leuA(G561R) 4.0 14067::p117_PCJ7-leuA (G561Y) 3.6

As shown in Table 5 above, it was confirmed that the L-leucineproductivity of the L-leucine-producing strains, Corynebacteriumglutamicum 14067::p117_PCJ7-leuA (R558A) and 14067::p117_PCJ7-leuA(R558Q), was improved 32- to 38-fold compared to the parent strain,Corynebacterium glutamicum ATCC14067.

Additionally, it was confirmed that the L-leucine productivity of theL-leucine-producing strains, Corynebacterium glutamicum14067::p117_PCJ7-leuA (G561R) and 14067::p117_PCJ7-leuA (G561Y), wasimproved about 36- to 40-fold compared to that of the parent strain,Corynebacterium glutamicum ATCC14067.

Based on the results above, it was confirmed the 558th and 561st aminoacids in the amino acid sequence (SEQ ID NO: 1) of isopropylmalatesynthase were important sites for the activity of the IPMS variantenzyme, and that even when each of the 558^(th) and 561^(st) amino acidsof the wild type IPMS protein was substituted with histidine andaspartic acid, respectively, the L-leucine productivity was remarkablyincreased in the strain having such modification.

While the present disclosure has been described with reference to theparticular illustrative embodiments, it will be understood by thoseskilled in the art to which the present disclosure pertains that thepresent disclosure may be embodied in other specific forms withoutdeparting from the technical spirit or essential characteristics of thepresent disclosure. Therefore, the embodiments described above areconsidered to be illustrative in all respects and not restrictive.Furthermore, the scope of the present disclosure is defined by theappended claims rather than the detailed description, and it should beunderstood that all modifications or variations derived from themeanings and scope of the present disclosure and equivalents thereof areincluded in the scope of the appended claims.

The invention claimed is:
 1. A modified polypeptide having anisopropylmalate synthase activity, wherein arginine at position 558 froma N-terminus of a polypeptide consisting of the amino acid sequence ofSEQ ID NO: 1 is substituted with histidine, alanine or glutamine; and/orglycine at position 561 from a N-terminus of a polypeptide consisting ofthe amino acid sequence of SEQ ID NO: 1 is substituted with arginine ortyrosine.
 2. The modified polypeptide according to claim 1, wherein themodified polypeptide consists of an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 21, 22, 23, 25, 26, 28, 29, 31, 32,34 and
 35. 3. A modified polypeptide having an isopropylmalate synthaseactivity, wherein arginine at position 558 from an N-terminus of apolypeptide consisting of the amino acid sequence of SEQ ID NO: 1 issubstituted with histidine, alanine, or glutamine, and glycine atposition 561 from an N-terminus of a polypeptide consisting of the aminoacid sequence of SEQ ID NO: 1 is substituted with aspartic acid,arginine, or tyrosine.
 4. The modified polypeptide according to claim 3,wherein the modified polypeptide consists of an amino acid sequenceselected from the group consisting of SEQ ID NOS: 27, 30, and
 33. 5. Apolynucleotide encoding the modified polypeptide of claim
 1. 6. Thepolynucleotide according to claim 5, wherein the polynucleotide consistsof a nucleotide sequence selected from the group consisting of SEQ IDNOS: 36, 37, 38, 40, 41, 43, 44, 46, 47, 49 and
 50. 7. A polynucleotideencoding the modified polypeptide of claim
 2. 8. A polynucleotideencoding the modified polypeptide of claim
 3. 9. The polynucleotideaccording to claim 8, wherein the polynucleotide consists of anucleotide sequence selected from the group consisting of SEQ ID NOS:42, 45, and
 48. 10. A microorganism of the genus Corynebacteriumproducing L-leucine, which is transformed with a vector comprising apolynucleotide encoding a modified polypeptide having an isopropylmalatesynthase activity, wherein arginine at position 558 from a N-terminus ofthe polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 issubstituted with an amino acid residue other than arginine; and/orglycine at position 561 from a N-terminus of the polypeptide consistingof the amino acid sequence of SEQ ID NO: 1 is substituted with an aminoacid residue other than glycine.
 11. A microorganism of the genusCorynebacterium producing L-leucine, comprising the modified polypeptideof claim
 1. 12. The microorganism according to claim 11, wherein themicroorganism of the genus Corynebacterium is Corynebacteriumglutamicum.
 13. A microorganism of the genus Corynebacterium producingL-leucine, comprising the modified polypeptide of claim
 2. 14. Amicroorganism of the genus Corynebacterium producing L-leucine,comprising the modified polypeptide of claim
 3. 15. A microorganism ofthe genus Corynebacterium producing L-leucine, comprising the modifiedpolypeptide of claim
 4. 16. A method of producing L-leucine, comprising:(a) culturing the microorganism of the genus Corynebacterium producingL-leucine according to claim 11 in a medium to produce L-leucine; and(b) recovering L-leucine from the cultured microorganism or the culturedmedium.
 17. A method of producing L-leucine, comprising: (a) culturingthe microorganism of the genus Corynebacterium producing L-leucineaccording to claim 13 in a medium to produce L-leucine; and (b)recovering L-leucine from the cultured microorganism or the culturedmedium.
 18. A method of producing L-leucine, comprising: (a) culturingthe microorganism of the genus Corynebacterium producing L-leucineaccording to claim 14 in a medium to produce L-leucine; and (b)recovering L-leucine from the cultured microorganism or the culturedmedium.
 19. A method of producing L-leucine, comprising: (a) culturingthe microorganism of the genus Corynebacterium producing L-leucineaccording to claim 15 in a medium to produce L-leucine; and (b)recovering L-leucine from the cultured microorganism or the culturedmedium.